Ii-Key/Her-2/neu hybrid cancer vaccine

ABSTRACT

Provided are methods and compositions for treating cancer in humans, the cancer being characterized by expression of Her-2/neu. The methods involve vaccinating a patient with an Ii-Key/MHC class II hybrid construct and thereby stimulating an immune response to the native Her-2/neu protein. The construct may be in the form of an Ii-Key hybrid peptide or a nucleic acid encoding an Ii-Key hybrid peptide. Methods are described wherein the cancer being treated is breast cancer. Also claimed is a pharmaceutical composition comprising an Ii-Key/MHC class II hybrid construct with and without an adjuvant. The adjuvant can include GM-CSF. The Ii-Key hybrid construct includes the LRMK (SEQ ID NO: 2) residues of Ii-Key protein and an MHC Class II epitope of a protein or portion thereof which is used in the vaccine or a DNA encoding the same hybrid peptide.

BACKGROUND OF THE INVENTION

Breast cancer is the most common cancer in women. Most breast cancersare detected early and treated with multimodality therapy includingsurgery, chemotherapy and radiation therapy. Despite patients beingrendered disease free with this intensive therapy, many women with highrisk features will have recurrent disease. Over expression of theHer-2/neu protein is one such high risk feature.

The epithelial cell adhesion molecule, Her-2/neu is a member of theepidermal growth factor receptor family, normally expressed during fetaldevelopment. Amplification of the gene and over-expression of theprotein product have been described in a number of epithelial tumors andare markers of high recurrence risk in breast cancer. Immunotherapydirected against Her-2/neu can control the growth of these tumors. TheHer-2/neu protein is over-expressed in 30-40% of early stage breastcancer and that over-expression is associated with poor clinicaloutcome. Other cancers in which Her-2/neu is over-expressed includeovary, recto-colon, lung, prostate, stomach, pancreatic, and biliarycancer (Sotiriadou, N. N., et al. Cancer Immunology Immunotherapy, inpress (online Sep. 8, 2006)).

The Her-2/neu protein is also a source of immunogenic peptides.Immunogenic peptides of Her-2/neu can stimulate cytotoxic T lymphocytes(CTL) to recognize and kill Her-2/neu expressing cancer cells in vitro.Some of the peptides (E75 and GP2) are being used in clinical trials asvaccines in patients with Her-2/neu+breast cancers. Thus far, they havebeen shown to be safe in patients and effective in stimulating antigenspecific immunity; more importantly, we have shown that the immunityconferred by E75 appears to have clinical benefit in preventingrecurrence of breast cancer. Unfortunately, the immunity conferred bythese peptide vaccines is not sustained. Helper peptides may be requiredto increase the efficiency of induction and establishment of long-termimmunity.

Helper peptides for Her-2/neu have been described. The MHC Class IIassociated Ii protein normally blocks the processing of endogenouspeptides, preventing attachment to MHC and antigen presentation.Suppression of the Ii protein in tumor cell lines and rat tumor modelshas been shown to induce tumor antigen presentation and enhance antigenspecific tumor cell killing.

The Ii protein normally binds to MHC class II molecules in theendoplasmic reticulum at synthesis and protects the epitope-binding siteon MHC class II molecules from binding to endogenously-derived epitopesin the endoplasmic reticulum, as normally occurs with MHC class Imolecules [Bertolino, P. and Rabourdin-Combe, C., Crit. Rev Immunol16:359-379 (1996); Bodmer, H., et al., Science 263:1284-1286 (1994)].Another major function of the Ii protein is to enhance exogenous peptidecharging to MHC class II molecules [Xu, M., et al., Mol Immunol31:723-731 (1994); Daibata, M., et al., Mol Immunol 31:255-260 (1994);Reyes, V. E., et al., Ann N Y Acad Sci 730:338-341 (1994)]. The MHCclass II/Ii complex is transported to a post-Golgi, antigenicpeptide-binding compartment after synthesis [Bakke, O. and DobbersteinB., Cell 63:707-716 (1990); Lamb, C. A. and Cresswell, P., J Immunol148:3478-3482 (1992); Blum, J. S, and Cresswell, P., Proc Natl Acad SciUSA 85:3975-3979 (1988)]. In such compartments, the Ii is cleaved byproteases to allow charging by exogenously derived epitopes. After beingcharged with epitopes, the MHC class II/epitope complex travels to thecell surface for presentation to CD4+ Th cells [Nguyen, Q. V et al., HumImmunol 24:153-163 (1989); Shi, G. P., et al., J Exp Med 191:1177-1186(2000); Riese, R. J., et al., Immunity 4:357-366 (1996); Hiltbold, E. M.and Roche, P. A., Curr Opin Immunol 14:30-35 (2002)]. Two mechanismshave been proposed to explain the function of Ii in enhancing thecharging of epitopes to MHC class II molecules. First, Ii is partiallydigested to leave only a small segment behind, termed CLIP, which isbound to the epitope-binding groove of the MHC class II molecule in amanner to keep the groove open [Riberdy, J. M., et al., Nature360:474-477 (1992); Gautam, A. M., et al., Proc Natl Acad Sci USA92:335-339 (1995); Romagnoli, P. and Germain, R. N., J Exp Med180:1107-1113 (1994)]. HLA-DM then exchanges CLIP for an epitope[Morris, P., et al., Nature 368:551-554 (1994); Denzin, L. K. andCresswell, P., Cell 82:155-165 (1995)]. Secondly, in a concerted manner,Ii is digested and released from MHC class II molecules as epitopes arebeing charged [Xu, M., et al., Mol Immunol 31:723-731 (1994); Daibata,M., et al., supra; Reyes, V. E., et al., Ann N Y Acad Sci 730:338-341(1994)].

An important function of the Ii protein is evident in a short sequencethat binds to an allosteric site outside of the epitope-binding groove[Xu, M., et al., Arzneimittelforschung 49:791-799 (1999)]. The result ofthis interaction is the maintenance of the epitope-binding groove in aconformation that is the most suitable for MHC class II molecules toexchange an epitope. That Ii sequence has been termed the Ii-Keypeptide. The segment of the Ii containing amino acids hIi(77-92)regulates tightness of closure of the antigenic epitope-binding grooveof MHC class II molecules. This segment first raised interest due toit's having 6 positive side chains, no negative side chains, and 4prolines, which together appeared to constitute a signal for a proteaseor “exchange-ase”; ostensibly to regulate cleavage and release of the Ii[Adams, S., et al., Arzneimittelforschung 47:1069-1077 (1997); Lu, S.,et al., J Immunol 145:899-904 (1990)]. Further studies showed thatmutations in this segment do, in fact, block the staged cleavage andrelease of Ii [Xu, M., et al., Mol Immunol 31:723-731 (1994); Daibata,M., et al., supra]. In light of these findings, we synthesized thefragment of the Ii containing amino acids hIi(77-92), referred to as“Ii-Key”. An initial study illustrated that the activation of hen eggwhite lysozyme (HEL)-specific T cell hybridoma by an HEL epitope peptidewas enhanced by Ii-Key peptides up to 50-fold [Adams, S., and Humphreys,R. E., Eur J Immunol 25:1693-1702 (1995)]. Enhanced activation wasobserved even when using paraformaldehyde-fixed Antigen Presenting Cells(APCs), in which normal intracellular processing was not possible.Studies to further identify the minimal active sequence of Ii-Keyrevealed a ‘core’ LRMKLPK structure that had greater potency than theoriginal 16-amino acid peptide [Adams, S., et al., supra]. The Ii 77-80(LRMK) segment retained at least 50% of the activity of LRMKLPK. Forsimplicity, we therefore designed later Ii-Key/MHC class II epitopehybrids with this shorter, four-amino-acid, Ii-Key moiety.

In contrast to cell culture studies, in vivo inoculation of mice withIi-Key plus an antigenic peptide failed to enhance the activity of thatantigenic peptide [unpublished observations]. This suggested that theIi-Key needed to be co-localized with the antigenic epitope to enhancepresentation. The Ii-Key moiety was linked covalently to the MHC classII epitope to ensure that individual MHC class II molecules on the APCare exposed simultaneously to both Ii-Key and epitope. A systematicseries of Ii-Key MHC class II epitope hybrids were synthesized andtested in an in vitro T cell hybridoma stimulation assay [Humphreys, R.E., et al., Vaccine 18:2693-2697 (2000)]. For this series of hybrids,the Ii-Key core (LRMK) was joined to an MHC class II-restricted epitopeof pigeon cytochrome C (PGCC81-104). The spacers joining Ii-Key andPGCC81-104 were either a simple polymethylene (δ-aminovaleric acid; ava)linker or the natural sequence of the Ii extending from the C-terminusof LRMK. The design of Ii-Key hybrids was based on biochemical and X-raycrystallographic data indicating that the Ii-Key binding site liesoutside of the antigenic peptide-binding groove of MHC class IImolecules [Ghosh P, et al., Nature 378:457-462 (1995)]. Both the lengthof the Ii-Key derivative and linker composition were varied within theseries. Hybrids having either type of bridge were effective. Somehybrids enhanced presentation of an antigenic epitope up to 250 timesabove the baseline stimulation observed using the free antigenic peptide[Humphreys, R. E. et al., supra].

The discovery of numerous clinically relevant peptide epitopes, both MHCclass I- and II-restricted, has increased the motivation to developeffective peptide vaccines. From these data, consensus motifs for bothMHC class I- and II-restricted epitopes have been proposed [Stevanovic,S, and Rammensee, H. G., Behring Inst Mitt: 7-13 (1994); Rammensee, H.G., et al., Immunogenetics 41:178-228 (1995); Hakenberg, J., et al.,Appl Bioinformatics 2:155-158 (2003)]. While some peptides of potentialclinical importance have been identified and specific immune responseshave been observed in patients treated with those peptides, goodtherapeutic efficacy has not been observed [Knutson, K. L., et al., ClinCancer Res 8:1014-1018 (2002); Phan, G. Q., et al., J Immunother26:349-356 (2003); Brinkman, J. A., et al., Expert Opin Biol Ther4:181-198 (2004); Hersey, P., et al., Cancer Immunol Immunother54:208-218 (2005)]. The main obstacle appears to be the relatively lowaffinity of some MHC class II-restricted epitopes. There is a need forpeptide vaccine potency to breakdown tolerance to tumor antigens.

A novel technology has been developed based on using a portion of the Iiprotein to enhance MHC class II epitope charging and thus the efficiencyof Th cell activation [Adams, S., and Humphreys, R. E., supra; Adams,S., et al., supra; Xu, M., et al., Arzneimittelforschung 49:791-799(1999); Xu, M., et al., Scand J Immunol 54:39-44 (2001)]. This “Ii-Key”segment of the Ii protein significantly enhances MHC class II epitopepresentation in a variety of settings and creates a practical method toenhance the efficacy of MHC class II peptide vaccines. The Ii-Keysegment binds an allosteric site on MHC class II molecules to loosentheir epitope-binding groove, allowing the epitope segment to directlycharge MHC class II molecules present on the cell surface [Adams, S., etal., supra; Xu, M., et al., Arzneimittelforschung 49:791-799 (1999)].Ii-Key hybrids are composed of the Ii-Key moiety linked to theN-terminus of an MHC class II epitope. This linkage can be of severalforms, including a simple polymethylene bridge or the natural sequenceof Ii extending from the C-terminus of LRMK or natural amino acids(extending from the N-terminal of the MHC class II epitope) of theprotein from which the MHC class II epitope peptide was obtained.

Ii-Key is a 4-amino-acid motif that has been reported to increase helperepitopes' occupancy of the MHC class II molecules and enhance CD4 T cellresponses. Ii-Key hybrids are much more potent than epitope-onlypeptides, both in vitro and in animal studies in vivo, when used inconjunction with epitopes relevant to different diseases [Humphreys, R.E. et al., supra; Gillogly, M. E., et al., Cancer Immunol Immunother53:490-496 (2004); Kallinteris, N. L., et al., Vaccine 21:4128-4132(2003); Kallinteris, N. L., et al., Vaccine 23:2336-2338 (2005);Kallinteris, N. L., et al., J Immunother 28:352-358 (2005)]. In vitro,some hybrids have been shown to enhance presentation of an antigenicepitope up to 250 times above the baseline stimulation observed usingthe free antigenic peptide [Humphreys, R. E., et al., supra]. Byenhancing the ability of peptide epitopes to charge MHC class IImolecules directly on the cell surface, Ii-Key hybrid technology opensthe door to a potent and clinically practical strategy for peptideimmunotherapy.

Another challenge specific to cancer immunotherapy is that tumorantigens are usually tolerated as self by the immune system. Therefore,the main task of clinical immunologists is to break down tolerance tospecific, tumor-associated self-antigens [Touloukian, C. E., et al., JImmunol 164:3535-3542 (2000); Knutson, K. L et al., supra; Phan, G. Q.,et al., supra; Brinkman, J. A., et al., supra; Hersey, P., et al.,supra]. We propose that the ability of Ii-Key hybrids to enhanceactivation of Th1 CD4+ cells will help greatly to break tolerance totumor antigens. In our in vitro and in vivo animal studies usingIi-Key/gp100 (46-58) and Ii-Key/HER-2/neu(777-789) hybrids [Kallinteris,N. L., et al., Vaccine 23:2336-2338 (2005); Kallinteris, N. L., et al.,Frontiers in Bioscience: 46-58 (2006)], significantly stronger CD4+ Tcell activity was obtained. Furthermore, the use of Ii-Key hybrids withinflammatory cytokines or adjuvants is expected to enhance the activityof hybrids and likewise help to breakdown tolerance against tumorantigens.

In order to assess the activity of Ii-Key hybrids in human cells, anIi-Key/HER-2/neu(777-789) epitope hybrid was used to stimulatelymphocytes from both a healthy donor and a patient with HER-2/neupositive metastatic breast carcinoma. The in vitro proliferation andInterferon (IFN)-γ release was more strongly stimulated by the Ii-Keyhybrid than by the epitope-only peptide [Gillogly, M. E et al., supra].Subsequent studies, using Peripheral Blood Mononuclear Cells (PBMC) frommore than 20 patients with HER-2/neu-positive cancer, have confirmed theincreased T helper activity of Ii-Key/HER-2/neu hybrids relative toepitope-only peptide in stimulating CTL effectors [Sotiriadou, N. N., etal. Cancer Immunology Immunotherapy, in press (online Sep. 8, 2006)].

Life threatening diseases such as cancer demand new efforts towardeffective vaccine design. Peptides represent a simple, safe, andadaptable basis for vaccine development. However, the potency of peptidevaccines is insufficient in most cases for significant therapeuticefficacy. The discovery of Ii-Key is of significant importance indesigning potent peptide vaccines. The MHC class II/epitope complex isrelatively stable [Fleckenstein, B., et al., Semin Immunol 11:405-416(1999); Joshi, R. V., et al., Biochemistry 39:3751-3762 (2000)]. Ii-Key(LRMK) facilitates the direct loading of epitopes to the MHC class IImolecule groove. Without Ii-Key, peptide epitopes have difficultydisplacing the pre-bound ambient peptides on MHC class II molecules atthe cell surface. With the help of Ii-Key, however, the peptide-bindinggroove on MHC class II molecules can be opened and closed easily,offering an efficient method to enhance the binding of vaccine peptidesto MHC class II molecules. Linking the Ii-Key moiety to an MHC class IIepitope, to generate an Ii-Key/MHC class II epitope hybrid, greatlyenhances the vaccine potency of the tethered epitope.

This type of vaccine development technology could greatly benefit tumorimmunotherapy. Peptides represent the safest form of all vaccinemodalities, as they are comprised of the minimal elements required forgeneration of an effective immune response: MHC class I and/or class IIepitopes. However, while immune responses have been observed usingpeptide vaccines, the demonstration of clinical efficacy is rare,pointing to the need for increased potency. Although peptide vaccineresearch initially focused on MHC class I epitopes to induce CTLactivity, MHC class II epitope vaccines for the induction of Th cellactivity have drawn growing attention [Wang, R. F., Immunol Rev188:65-80 (2002); Sette A., and Fikes, J., Curr Opin Immunol 15:461-470(2003); Hanson, H. L. et al., J Immunol 172:4215-4224 (2004); Wong, R.,et al., Clin Cancer Res 10:5004-5013 (2004)]. Recent data has clearlyshown that CD4+ Th cell activation is required for the induction of apotent immune response against an immunogen. Antigen-specific Th cellsare needed for full activation of antigen-specific CD8+ CTLs and toprovide long-term antigen-specific memory [Hung, K., et al., J Exp Med188:2357-2368 (1998); Surman, D. R., et al., J Immunol 164:562-565(2000); Welsh, R. M., et al., Annu Rev Immunol 22:711-743 (2004)].

CD4+ Th cells play a critical role by inducing and maintaining both CD8+T cell and B cell responses and in maintaining immunological memory[Hung, K., et al., J Exp Med 188:2357-2368 (1998); Surman, D. R., etal., J Immunol 164:562-565 (2000); Welsh, R. M., et al., Annu RevImmunol 22:711-743 (2004), Knutson, K. L. and Disis, M. L., CancerImmunol Immunother (2005); Rocha B. and Tanchot, C., Curr Opin Immunol16:259-263 (2004); Janssen, E. M., et al., Nature 434:88-93 (2005);Dissanayake, S. K., et al., Cancer Res 64:1867-1874 (2004)]. Forexample, F. Ossendorp et al. established that tumor antigen-specific Thcells are required for optimal induction of CTLs against MHC classII-negative tumors [J Exp Med 187:693-702 (1998)]. The role of CD4+ Thcells in cancer immunity is further highlighted by significant clinicalresults obtained in melanoma patients receiving adoptive transfer ofhighly reactive CD8+ and CD4+ T cells [Dudley, M. E., et al., Science298:850-854 (2002); Robbins, P. F., et al., J Immunol 169:6036-6047(2002)]. F. G. Gao et al. showed that in order to activate memory CD8+ Tcells to become fully functional tumor killer cells, antigen-specificCD4+ Th cells were required [Cancer Res 62:6438-6441 (2002)]. P. Yu etal. defined how the complementary role of CD4+ Th cells is required forefficient cross-presentation of tumor antigens to CD8+ T cells [J ExpMed 197:985-995 (2003)]. Furthermore, CD4+ T cells can help to breakdown tolerance to persistent self-antigens (e.g., tumor-associatedantigens) to fight established tumors in an Interleukin (IL)-2 dependentmechanism [Anthony, P. A., et al., J Immunol 174:2591-2601 (2005)].Along with the continuing discovery of novel defined epitopes, theinvestigation of MHC class II epitope-based vaccines in tumorimmunotherapy is advancing [Wang, R. F., supra; Sette A., and Fikes, J.,supra; Hanson, H. L. et al., supra; Wong, R., et al., supra; Mandic, M.,et al., J Immunol 174:1751-1759 (2005); Lu, J., et al., J Immunol172:4575-4582 (2004); Slager, E. H., et al., J Immunol 172:5095-5102(2004)].

Conventional peptide vaccines have a number of disadvantages. First,they do not stimulate CD4+ T lymphocyte responses, which leads to a lackof B cell response and a lack of immunologic memory. Second, they arenot well processed by the immune system. Therefore, clinically, thesevaccines require coadministration with immune stimulants such asGranulocyte Monocyte-Colony Stimulating Factor (GM-CSF). Moreimportantly, the vaccines are of limited use, restricted to only thosepatients with HLA A-2 subtype, an HLA phenotype which occurs only inabout 50% of the population but which most efficiently processes thesepeptides for antigen expression.

Several methods such as LEAPS and ISCOMATRIX have been developed toenhance the potency of peptide vaccines. A variety of techniques havebeen explored to improve the activity of peptide vaccines but none areMHC class II epitope-specific. These methods use different mechanismsfor peptide vaccine enhancement. Improving the delivery of MHC class IIepitopes into APCs is a common approach to enhancing MHC class IIpeptide vaccines. For example, exosomes are used as a delivery vehiclefor better activation of both CD8+ and CD4+ T cells [Delcayre, A. and LePecq, J. B., Opin Mol. Ther. 8:31-8 (2006)]. Another method isISCOMATRIX which is a cage-like structure composed of antigens, such aspeptides, and adjuvants [Sanders, M. T., et al., Immunol Cell Biol.83:119-28 (2005)]. ISCOMATRIX effectively induces both humoral andcellular immune responses against the antigens incorporated in thestructure by enhancing the delivery of peptide antigens and by providingadjuvant stimulation. APCs usually have difficulty acquiring solubleantigen. Antigen/antibody complexes are more accessible to APCs throughthe recognition of the Fcγ receptor on APCs by the Fc domain on anantibody [Nagata, Y., et al., Proc Natl Acad Sci USA 99:10629-34(2002)]. Molecular chaperones are necessary components for betterbinding of epitopes to MHC class II molecules. Bacterial HSP 70 enhancesthe immune response against MHC class II epitopes complexed to bacterialHSP70 [Tobian, A. et al., J Immunol 172:5277-86 (2004)]. However, theenhancement occurs only at low pH, indicating that chaperone-complexedepitope binding to MHC class II is also limited by CLIP [Urban, R. G.,et al., J Exp Med 180:751-755 (1994)]. More recently, peptide conjugatestargeting specific components of immune cells have been investigated. Anexample of the latter employs a T cell-binding ligand coupled to apeptide antigen (Ligand Epitope Antigen Presentation System, LEAPS)[Zimmerman, D. H. and Rosenthal, K. S., Front Biosci 10:790-798 (2005)].Because Ii-Key hybrids target the charging of MHC class II molecules,antigen presentation to T cells is more selective than LEAPS technology.Peptide vaccines are usually limited by polymorphic MHC class II allelerestrictions. A non-specific T helper epitope technology, the pan-DRepitope (PADRE), has also been developed to circumvent this limitation[Franke, E. D., et al., Vaccine 17:1201-5 (1999)]. The advantage ofPADRE is that it overcomes restrictions by HLA-DR alleles. DNA vaccinesusing the Ii gene as a delivery carrier to deliver T helper epitope toMHC class II molecules have been developed [Nagata, T et al., Vaccine20:105-14 (2001)]. This method utilizes an Ii gene in which the CLIPportion has been replaced by a DNA fragment encoding a MHC class IIepitope. This method has successfully induced epitope-specific CD4+ Tcells. It should also be noted that Ii-Key hybrid technology iscompatible with many of the methods discussed to further enhance theoverall antigen-specific response. For example, Ii-Key hybrid peptidesmight be incorporated into ISCOMATRIX to further enhance the potency ofthe vaccine.

A hybrid peptide of a Her-2/neu antigenic epitope p776-790 and theIi-Key protein has been developed. This hybrid protein, called AE37,brings the antigenic epitope into close proximity of the target bindingsite on class II MHC molecules, thus priming the molecule for antigenpresentation. We have performed a phase Ib trial of the AE37 peptidevaccine in human Her-2/neu+ breast cancer patients to document toxicityand measure immunologic responses to escalating doses of the vaccine.The results of this trial are presented here.

SUMMARY OF THE INVENTION

In one aspect, the present invention involves methods to treat cancer inhumans, the cancer being characterized by expression of the Her-2/neuprotein. The methods include the treatment of breast cancer. The methodsentail providing an Ii-Key/MHC class II hybrid construct and stimulatinga patient's immune system by vaccination with the hybrid construct.Vaccination with the hybrid construct stimulates CD 4+ T cell activationand supports a CTL response specific to the native Her-2/neu protein.

In another aspect, the invention involves pharmaceutical compositionsfor use in the treatment of cancer. A composition of the inventioncomprises the hybrid construct in a pharmaceutically acceptable carrier.The hybrid construct includes the LRMK residues of the Ii protein linkedto the N-terminus of an MHC class II epitope of Her-2/neu or a DNAencoding the same peptide. The Her-2/neu epitope included in theconstruct may be GVGSPYVSRLLGICL. The composition may further comprisean adjuvant which may be GM-CSF.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-4: Diagrams represent the local DTH reaction (solid lines) anddose of GM-CSF (dashed lines). Patients were vaccinated monthly, for sixmonths, with peptide plus GM-CSF in different dose groups. Each patientwas evaluated 48-72 hours later for local and systemic toxicity. A localreaction exceeding 100 mm induration necessitated a dose reduction inthe GM-CSF for subsequent vaccinations.

FIG. 1: Patients were vaccinated with 100 μg AE37 peptide plus 250 μgGM-CSF, monthly, for six months.

FIG. 2: Patients were vaccinated with 500 μg AE37 peptide plus 250 μgGM-CSF initially. A GM-CSF reduction was necessary in all patients bydose R3.

FIG. 3: Patients were initially vaccinated with 1000 μg AE37 peptidewithout GM-CSF. Patient A9V9 was given an increased dose of GM-CSFbeginning at dose R4.

FIG. 4: The same patients from FIG. 3 were initially vaccinated with1000 μg AE37 peptide. Patients A7V7 and A8V8 were given a decreased doseof AE37 by dose R4.

FIG. 5: Delayed Type Hypersensitivity (DTH). Patients were inoculatedwith 100 μg AE37 peptide without GM-CSF prior (pre) to the initiation ofand one month after (post) completion of the six dose vaccine schedule.Local reaction to AE37 was measured (mean diameter of the induration)48-72 hours later.

FIG. 6: DTH by dosing groups. The pre- and post-vaccination meanreaction of each dosing group is shown. The x-axis labels describe thedosing regimen. 100:6=100 μg AE37 plus 250 μg GM-CSF, six doses;500:6=500 μg AE37 plus 250 μg GM-CSF initially, six doses;1000:6=initially 1000 μg AE37 without GM-CSF, six doses; 500:125=500 μgAE37 plus 125 μg GM-CSF, six doses.

FIGS. 7-10: Ex Vivo Interferon-γ Elispot. PBMC's were drawn frompatients before (pre) the first inoculation, before each subsequentmonthly inoculation, and one month after the series ended (post). PBMC'swere stimulated with peptide and IFN-γ activity was measured. Thediagram represents the number of spots per million PBMC's before,during, and after the vaccination series. “Max” is the highest of thesix measurements taken before each inoculation in the series.

FIG. 7: PBMC's were stimulated with 2.5 μg AE37 peptide.

FIG. 8: PBMC's were stimulated with 25 μg AE37 peptide.

FIG. 9: PBMC's were stimulated with 2.5 μg AE36 peptide.

FIG. 10: PBMC's were stimulated with 25 μg AE36 peptide.

FIGS. 11-14: 7 Day Elispot. PBMC's were drawn from patients before (pre)the first inoculation, before each subsequent monthly inoculation, andone month after the series ended (post). PBMC's were stimulated andallowed to incubate with peptide for seven days before CTL-ImmunoSpotAnalysis. The diagram represents the number of spots per million PBMC'sbefore, during, and after the vaccination series. “Max” is the highestof the six measurements taken before each inoculation in the series.

FIG. 11: PBMC's were stimulated with 1.0 μg AE37 peptide.

FIG. 12: PBMC's were stimulated with 1.0 μg AE36 peptide.

FIG. 13: PBMC's were stimulated with 10 μg AE37 peptide.

FIG. 14: PBMC's were stimulated with 10 μg AE36 peptide.

FIGS. 15-18: Proliferation of T cells. PBMC's were drawn from patientsbefore (pre) the first inoculation, before each subsequent monthlyinoculation, one month after the series ended (post), and six monthsafter the series ended (memory). PBMC's stimulated and incubated withpeptide were pulsed with radioactive ³H-thymidine and counted with ascintillation counter. The amount of ³H-thymidine incorporated isassociated with the peptide-specific proliferative activity of thecultures.

FIG. 15: PBMC's were stimulated with 1.0 μg AE37 peptide.

FIG. 16: PBMC's were stimulated with 1.0 μg AE36 peptide.

FIG. 17: PBMC's were stimulated with 10 μg AE37 peptide.

FIG. 18: PBMC's were stimulated with 10 μg AE36 peptide.

DETAILED DESCRIPTION OF THE INVENTION

The hybrid vaccine composition and methods of use disclosed herein havebeen designed to overcome the shortcomings of conventional peptidevaccines. By taking advantage of the Ii-Key protein interaction withClass II MHC molecules, the compositions and methods of the presentinvention bring the antigenic epitope of Her-2/neu to the Class II MHCbinding groove, bypassing the normal antigen processing pathway. In thiscontext, antigen can then be presented to the immune system, stimulatinga specific CD4+ T lymphocyte response. Due to the increased potency ofIi-Key/MHC class II hybrids in stimulating immune response, lessefficiency in the process can be tolerated, and use of the vaccine doesnot have to be limited to HLA A-2 patients, and it may not require theuse of immune system stimulants.

Described herein is a method of treating a cancer in humans, the cancerbeing characterized by expression of Her-2/neu. As noted in theBackground section, cancers which express Her-2/neu include breast,ovary, recto-colon, lung, prostate, stomach, pancreatic, and biliarycancers. The method comprises providing an Ii-Key/MHC class II hybridconstruct in a pharmaceutically acceptable carrier and vaccinating apatient with the hybrid, under conditions appropriate for thestimulation of an immune response. The hybrid construct may beadministered as an Ii-Key hybrid peptide or in the form of a nucleicacid encoding an Ii-Key hybrid peptide. The Ii-Key/MHC class II hybridpeptide comprises the LRMK amino acid residues of the Ii protein linkedto the N-terminus of an MHC class II epitope containing segment ofHer-2/neu. The LRMK residues and the MHC class II epitope should beseparated by a distance equivalent to the length of about two to twentyamino acid residues. This space can contain a variety of linkers,including a simple polymethylene (ava) linker, the natural sequence ofIi extending from the C-terminus of LRMK, or the natural sequence ofHer-2/neu extending from the N-terminus of the MHC class II epitope. Themethod results in the stimulation of a CD4+ T cell response. Similarly,the hybrid construct can be administered in the form of a nucleic acidencoding an Ii-Key/Her-2/neu hybrid peptide.

As is detailed in the Exemplification section, human cancer patientswere vaccinated with AE37, a hybrid Ii-Key/Her-2/neu MHC class IIpeptide. PBMC's were drawn from the patients and analyzed using IFN-γELISPOT and peptide-specific Proliferation assays. The ELISPOT analysisshowed (see FIGS. 9, 10, 12, and 14) that patients exhibited anincreased CD4+ T cell response to AE36, the native Her-2/neu MHC classII peptide, after being vaccinated with the Ii-Key/Her-2/neu hybridpeptide. The Proliferation Assay showed that CD4+ T cells had increasedcapacity to divide and expand a specific clone that recognized thenative Her-2/neu MHC class II peptide (see FIGS. 16 and 18).

The method of the present invention includes treating a cancer patientby vaccination with an Ii-Key/Her-2/neu hybrid construct whereby thestimulation of a CD4+ T cell response specific to native Her-2/neupeptide is enhanced. The ELISPOT and Proliferation assays show increasedCD4+ T cell response to the native Her-2/neu peptide but not to thenon-Her-2/neu negative control peptide AEN (data not shown) in patientsvaccinated with the Ii-Key/Her-2/neu hybrid construct.

The method of the present invention includes treating a cancer patientby vaccination with an Ii-Key/MHC class II hybrid construct wherein theMHC class II epitope is contained within the peptide GVGSPYVSRLLGICL.The method comprises treating a cancer patient by vaccination with ahybrid peptide with the amino acid sequence LRMKGVGSPYVSRLLGICL or byvaccination with a DNA encoding the same. The Patient characteristicsand clinical protocols section of the Exemplification discloses theamino acid sequences used for the MHC class II epitope containingsegment and the peptide used in the AE37 vaccine.

The patients used in this study were breast cancer patients, thereforethe method of the invention includes the treatment of breast cancerpatients by vaccination with an Ii-Key/Her-2/neu hybrid peptide or witha DNA encoding the same. Since other cancers are known to expressHer-2/neu, it will be readily apparent to a person having ordinary skillin the art that the preceding method may be implemented in the treatmentof not only breast cancer but of any cancer which expresses Her-2/neu.Thus the invention includes methods of treating ovary, recto-colon,lung, prostate, stomach, pancreatic, or biliary cancers by vaccinationwith an Ii-Key/Her-2/neu hybrid peptide.

In another embodiment, the present invention provides a pharmaceuticalcomposition, for use in the treatment of cancer, comprising anIi-Key/MHC Class II hybrid construct in a pharmaceutically acceptablecarrier. A critical requirement of a pharmaceutical composition intendedfor use in humans is safety. The Exemplification Section includesexperiments demonstrating safety in humans. The Ii-Key/MHC Class IIhybrid construct comprises the LRMK residues of the Ii protein linked tothe N-terminus of an MHC Class II epitope-containing segment ofHer-2/neu. The LRMK residues and the MHC class II epitope should beseparated by a distance equivalent to the length of about two to twentyamino acid residues. This space can contain a variety of linkers,including a simple polymethylene (ava) linker, the natural sequence ofIi extending from the C-terminus of LRMK, or the natural sequence ofHer-2/neu extending from the N-terminus of the MHC class II epitope. TheIi-Key/MHC Class II hybrid construct can also comprise a DNA encodingthe same hybrid peptide. More specifically the present inventionincludes a composition wherein the MHC Class II epitope of the hybridconstruct is contained within the peptide GVGSPYVSRLLGICL. Thecomposition of the present invention includes the hybrid constructcomprising amino acids having the sequence LRMKGVGSPYVSRLLGICL or a DNAencoding the same. The results in the Exemplification of the ELISPOT andProliferation assays show that human cancer patients vaccinated withIi-Key/Her-2/neu hybrid constructs exhibit an increased CD4+ T cellresponse to native Her-2/neu peptide.

The present invention further provides a pharmaceutical composition, foruse in the treatment of cancer, comprising an adjuvant and an Ii-Key/MHCClass II hybrid construct in a pharmaceutically acceptable carrier. TheIi-Key/MHC Class II hybrid construct comprises the LRMK residues ofIi-Key protein linked to the N-terminus of an MHC Class II epitopecontaining segment of Her-2/neu. The construct can also comprise a DNAencoding the same hybrid peptide. The composition provided may includethe adjuvant GM-CSF. The Vaccine and Vaccination Series sections of theExemplification and FIGS. 1-4 describe the inclusion of an adjuvant,GM-CSF, in the hybrid construct compositions administered to the cancerpatients in the clinical trial. The ELISPOT and peptide-specificProliferation assays disclose that patients receiving a compositioncomprising an Ii-Key/Her-2/neu hybrid construct and an adjuvant showincreased immunologic responses.

Ii-Key/MHC class II hybrid vaccines can induce long-term,antigen-specific CD4+ T cell stimulation. The enhanced Th cellactivation afforded by hybridization with Ii-Key represents an importantadvance in the design of peptide vaccines. Furthermore, theantigen-specific mechanism of T helper stimulation allows Ii-Key hybridtechnology to be used together with other strategies (such asISCOMATRIX) to further enhance the potency of the MHC class II vaccinepeptides.

It will be recognized by one skilled in the art that the hybridconstruct composition may be administered in the form of an Ii-Keyhybrid peptide or as a nucleic acid construct encoding anamino-acid-based Ii-Key hybrid peptide. One skilled in the art, usingroutine experimental methods, could also substitute various natural ornon-natural amino acids at respective residue positions in the hybridpeptide. Some examples of molecules which may be substituted arepeptidomimetic structures, D-isomer amino acids, N-methyl amino acids,L-isomer amino acids, modified L-isomer amino acids, and cyclizedderivatives.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of theinvention.

EXEMPLIFICATION Patient Characteristics and Clinical Protocols

The Walter Reed Army Medical Center Department of Clinical Investigationapproved the clinical protocol. This clinical trial is being conductedunder an investigational new drug application (IND12229) approved by theFood and Drug Administration. All patients had histologically confirmednode negative breast cancer (NNBC) that expressed HER-2/neu by standardimmunohistochemistry. All patients had completed a standard course ofsurgery, chemotherapy, and radiation therapy (as required) beforeenrollment, and those patients on chemoprevention continued on theirspecific regimens. After screening for eligibility criteria and propercounseling and consenting, patients with NNBC were enrolled into thestudy and HLA typed. HLA A2-patients and HLA A2+ patients who were notinterested in an alternative vaccine trial were vaccinated. Beforevaccination, patients were skin tested with a panel of recall antigens(Mantoux test: mumps, tetanus, and Candida). Patients reacting (>5 mminduration) to at least two antigens were considered immunocompetent.

To investigate the safety of the AE37 hybrid vaccine, we have thus farenrolled 15 patients with NNBC who were disease-free after standardtherapy. The dose escalation safety trial design is for five groups ofthree patients to receive escalating doeses of AE37 with or withoutGM-CSF in six monthly inoculations. An additional six patients will bevaccinated at the optimal biologic dosing (OBD) in order to complete the21 patient phase I trial. Enrolled patients were HLA typed, and HLA-A2⁻patients and those HLA-A2⁺ patients not interested in an alternativevaccine trial were vaccinated. Table 2 shows patient demographics.

TABLE 2 Patient demographics (n = 15 unless specified) Median age, years56 Range, years 44-70 Race White 60 Black 33 Asian  7 Tumor size* T1, %89 T2-T4, % 11 Histological grade* I-II, % 67 III, % 33 ER-negative,PR-negative, %* 22 No chemotherapy, % 80 No XRT, % 23 No hormonaltherapy, % 20 *n = 18 (total number of tumors in 15 patients); ER =Estrogen Receptor; PR = Progesterone Receptor

Vaccine

The Ii-Key/HER-2/neu MHC class II Peptide, AE37(Ac-LRMKGVGSPYVSRLLGICL-NH₂) was commercially produced in goodmanufacturing practices (GMP) grade by NeoMPS Inc (San Diego, Calif.).Peptide purity was verified by high-performance liquid chromatographyand mass spectrometry, and the amino acid content was determined byamino acid analysis. The peptide was purified to more than 95%.Sterility and general safety testing was carried out by the manufacturerand conformed to FDA requirements. Lyophilized peptide was reconstitutedin sterile saline at the following concentrations: 100 μg in 0.5 ml; 500μg in 0.5 ml; and 1 mg in 0.5 ml. Vaccine was mixed with GM-CSF (Berlex,Seattle, Wash.) at varying concentrations (see below) in 0.5 ml. The1.0-ml inoculation was divided and given intradermally at two sites(split injection) within 5 cm of each other. All inoculations were givenin the same extremity.

Vaccination Series

The study was performed as a two-stage safety trial to define themaximum tolerated dose of vaccine as well as the optimal dosing of thevaccine and GM-CSF. In the first stage (FIGS. 1-4), patient dose groupswere given escalating amounts of the AE37 peptide. Three patients wereassigned to each dosing group and were given the assigned dose of AE37with a fixed initial dose of GM-CSF of 250 μg. This dose of GM-CSF waschosen based on our previous E75 trials. However, GM-CSF was reduced 50%for subsequent inoculations when local skin reaction exceeded 100 mm.All patients received monthly inoculations for six months. Dosing groupswere as follows (AE37 (μg):#inoculations): 100:6, 500:6, 1000:6. Becauseeach patient in the 500:6 group required significant and repeatedreductions of GM-CSF, the 1000:6 group was initially inoculated withoutGM-CSF; GM-CSF was added back if local reaction from the two injectionsites were non-confluent.

In the second stage of the trial, optimal dosing of GM-CSF was assessedwith a fixed dose of AE37. Given the dose reductions required with the1000:6 dose group, we chose 500 μg as the fixed AE37 dose. Threeadditional groups of three patients each were inoculated with 500 μgvaccine with decreasing initial doses of GM-CSF to determine the optimalcombination dosing. These groups were as follows (AE37 (μg):initialGM-CSF dose (μg)): 500:125, 500:30, and 500:TBD. If the 500:30 dose istolerated for the complete series and produces a good local reaction inall three patients, then this dose will be the optimal biologic dose(OBD) and an additional three patients will be inoculated with thisregimen. However, if any patient requires a dose escalation of theGM-CSF dose, then an additional group will be added, 500:62.5.Alternatively, if any patient does not tolerate the 500:30 and requiresremoval of the GM-CSF, then an additional group will be added, 500:0.

TABLE 1 Dosing of vaccine and adjuvant AE37 GM-CSF dose initial doseDose Group Patient number (μg) (μg)* Schedule Stage I  100:6 A1, A2, A3100 250 Monthly × 6  500:6 A4, A5, A6 500 250 Monthly × 6 1000:6 A7, A8,A9 1000 0 Monthly × 6 Stage II  500:125 A10, A11, A12 500 125 Monthly ×6  500:30 A13, A14, A15 500 30 Monthly × 6 (A16-18 if OBD) 500 Monthly ×6 Possible  500:62.5 (A16-18) 500 62.5 Monthly × 6  500:0 (A16-18) 500 0Monthly × 6 *If patient's local reaction measured >100 mm, then GM-CSF(or peptide if no GM-CSF given) dose was reduced 50%.

The hybrid peptide AE37 was mixed with GM-CSF (varying doses) andinjected intradermally in the same extremity on a monthly basis. Eachdosing group consisted of 3 patients. Patients were enrolledsequentially; however, if a patient failed to complete a series, areplacement patient was given the same dose until each group wascomplete. Table 1 provides the two-stage safety and dose-escalationschedule. Stage I was designed to identify the maximum tolerated dose ofvaccine. Stage II was designed to find the optimal biologic dose. Localtoxicity dictated dose adjustments in each stage.

A local reaction ≧100 mm in at least one direction necessitates areduction of GM-CSF dose for the subsequent vaccination (or reduction invaccine dose if patient received no GM-CSF). The first group (100:6)required no reductions in dose. However, each patient in the secondgroup (500:6) required a GM-CSF dose reduction by the third vaccine.FIGS. 1-4 show the dose of GM-CSF and the local reaction in each patientthroughout the vaccine series. Given the robust local reactions, thethird group (1000:6) was initiated without any GM-CSF; two of the threepatients in this group required reduction of vaccine dose on multipleoccasions. The third patient in this group did not show robust localresponse and GM-CSF was added back to her vaccine schedule in anescalating fashion.

Based on the findings in the first stage, stage II was initiated withpatients scheduled to receive the vaccine at a fixed dose (AE37=500 μg)with different doses of adjuvant to find the optimal combination. Dosereductions of GM-CSF were again dictated by a local reaction in anydimension ≧100 mm.

Toxicity

The National Institutes of Health Common Toxicity Criteria (CTC)(Version 2, Mar. 23, 1998) definitions of adverse events were applied.Both local toxicity at the injection sites as well as systemic toxicitywere evaluated in all patients for all inoculations. By design,progression to the second stage occurred only if no significant toxicityoccurred in the first stage.

After each vaccination, the patients were observed for 1 hour for signsof a hypersensitivity reaction; they returned 48-72 hours later to bequestioned regarding local and systemic reactions and have theirinjection sites checked. Toxicities were graded per the NationalInstitutes of Health Common Toxicity Criteria and reported on a scalezero to five (Table 3). Local toxicity dictated dose reductions asdescribed above. All patients had grade 1 or 2 local toxicity (desiredeffect) and no grade 3 or greater local toxicities have been reported.Only grade 1 systemic toxicities were reported. So far, 12 patients havecompleted the vaccine series and their have been no patient withdrawalsfrom the trial.

TABLE 3 Maximum Toxicity Local Systemic Toxicity Toxicity Patient Grade1 Grade 2 Grade 3 Grade 1 Grade 2 Grade 3 Group 1 1 2 0  3 0 0 100 μgAE37 + 250 μg GM-CSF Group 2 0 3 0  2 0 0 500 μg AE37 + 250 μg GM-CSFGroup 3 1 2 0  2 0 0 1000 μg AE37 Group 4 1 2 0  3 0 0 500 μg AE37 + 125μg GM-CSF TOTAL 3(25)% 9(75%) 0(0%) 10(83%) 0(0%) 0(0%)

Control Peptides and Proteins

The AE37 peptide used in this study is a fusion/hybrid of the Ii-Keypeptide (LRMK) with the native HER-2/neu peptide (aa776-790:GVGSPYVSRLLGICL). In order to assess/determine that immune responsesbeing measured/monitored are representative/indicative of reactivityagainst the native HER-2/neu peptide, we synthesized the native peptide,Her-2/neu MHC class II Peptide, AE36 (Ac-GVGSPYVSRLLGICL-NH₂). Inaddition, a negative control peptide, AEN, containing the Ii-Key peptidefused to a non-HER-2/neu sequence (Ac-LRMK-ava-YVDRFYKTLRAEQ-NH₂) wasalso used in some of the immune assays. The non-HER-2/neu sequence isthat of a promiscuous class II peptide from the HIV gag protein. TetanusToxoid (TT) (List Biologicals Inc) was used as a positive controlantigen for the immune assays.

Peripheral Blood Mononuclear Cell (PBMC) Isolation and Cultures

Blood was drawn from patients prior to receiving each inoculation, atone month (post-vaccination), and at six months (long-term) aftercompleting the series. Forty ml of peripheral blood was drawn intoVacutainer® CPT™ tubes (Becton Dickinson, Franklin Lakes, N.J.) andcentrifuged for the isolation of PBMC populations. PBMC were washed inHBSS and re-suspended in culture medium (CM) consisting of RPMI-1640containing 10% FCS (Gemini Bio-Products, Woodland, Calif.) supplementedwith 1× penicillin/L-glutamine/streptomycin (Life Technologies,Gaithersburg, Md.) and used for setting up the immunomonitoringprotocol/assays which consisted of ELISPOT (IFN-γ secretion) andproliferation (3H-thymidine incorporation) assays. All cultures weremaintained in a humidified incubator at 37° C. in 5% CO₂.

IFN-γ ELISPOT Assays

ELISPOT assays measure the cytokine secretion of T cells. In order tomonitor the ongoing immune response in the patients receiving/to theAE37 vaccination we measured the IFN-γ secretion activity in PBMCcultures stimulated with culture medium (CM), AE36, AE37, AEN or TTusing the ELISPOT assay. The ELISPOT assay was set up as a direct or exvivo ELISPOT assay and a 7-day ELISPOT assay that has been developed inour laboratory.

Ex vivo IFN-γ-+ELISPOT assay: The human IFN-γ ELISPOT Kit(BD-Pharmingen) was used for the ELISPOT assays. 100 μl aliquots of CMcontaining 40 ng/ml of recombinant human IL-7 (R&D Systems, MN) wereadded to 8 wells of an ELISPOT plate. The plate was placed in a CO₂incubator for a period of at least 30 minutes. During this time the PBMCpopulation prepared from the patient's peripheral blood (as described inthe previous section) was resuspended at 5×10⁶ cells/ml in CM and addedas 100 μl aliquots to 8 polystyrene tubes (BD-Falcon, NJ). Each peptide(AE36 or AE37 or AEN) or antigen (TT) was added to a separate tube whileone tube had no stimulant added to it and this served as the CM controltube. The AE37 and control peptides and antigen were added in thefollowing amounts: 2.5 μg AEN, 25 μg AEN, 2.5 μg AE36, 25 μg AE36, 2.5μg AE37, 25 μg AE37, 0.2 μg TT. The aliquots of cell suspensions withthe added peptides/antigen were mixed thoroughly and transferred to therespective/separate wells of the ELISPOT plate containing the CM+IL-7that had been placed earlier in the incubator. The plate was thenreturned to the incubator for an overnight incubation of approximately16-18 hours. On the next day the plate was developed as permanufacturers' instructions in the kit. The plate was allowed to dry andthe number of spots present in each well was counted and analyzed usinga CTL-ImmunoSpot Analyzer (C.T.L. Cellular Technology Ltd, OH) (FIGS.7-10). The full range of concentrations and peptides tested for eachpatient varied depending upon the availability of sufficient PBMC yieldfrom the patient's blood sample. Whenever possible the assay was set upin duplicates.

7-day IFN-γ ELISPOT assay: As a method of increasing or expanding thesensitivity and capability of detecting the presence and activity ofvaccine-specific T cells being induced in the patients by the AE37vaccine, we have developed a modified ELISPOT assay that is performedusing cells from 7-day cultures that have been stimulated in the absenceor presence of the same panel of peptides used in the ex vivo ELISPOTassay. Briefly, each peptide (AE36, AE37 or AEN) or antigen (TT) wasadded to a separate well of a 48-well plate while one well had nostimulant added to it and this served as the CM control well. The AE37and control peptides and antigen were added in the following amounts: 1μg AEN, 10 μg AEN, 1 μg AE36, 10 μg AE36, 1 μg AE37, 10 μg AE37, 1 μgTT. The PBMC population prepared from the patient's peripheral blood (asdescribed in the previous section) was resuspended at 2×10⁶ cells/ml inCM and added as 1 ml aliquots to the 8 wells. The plate was thenincubated in a humidified CO₂ incubator for a period of 7 days. At theend of the incubation period the plate was removed and thecells/cultures were harvested into separate tubes, spun down,resuspended in CM, and counted. The separate populations of cells werethen added at 1×10⁵, 2×10⁵, or 3×10⁵ cells/100 μl to separate/respectivewells of an ELISPOT plate that had been pre-incubated with 100 μl perwell of CM (without IL-7) for a minimal time period of 30 minutes. Theplate was then returned to the incubator for an overnight incubation ofapproximately 16-18 hours. On the next day the plate was developed asper manufacturers' instructions in the kit. The plate was allowed to dryand the number of spots present in each well was counted and analyzedusing a CTL-ImmunoSpot Analyzer (C.T.L. Cellular Technology Ltd, OH)(FIGS. 11-14). The full range of concentrations and peptides and numbersof cells/well tested for each patient varied depending upon theavailability of sufficient cells recovered from the 7-day cultures.Whenever possible the assay was set up in duplicates.

Proliferation Assay

The PBMC population prepared from the patient's peripheral blood samplewas also used for the monitoring of vaccine-specific proliferativeactivity of the T lymphocytes using a standard radioactive ³H-thymidineincorporation assay. The proliferation assay measures a T cell'scapacity to divide and thus expand a specific clone that recognizes thestimulating antigen. Briefly, the PBMC population was stimulated in theabsence or presence of the same panel of peptides used in the ex vivoELISPOT assay. Each peptide (AE36 or AE37 or AEN) or antigen (TT) wasadded to 3 separate wells (triplicates) of a 96-round bottom well platewhile one set of wells had no stimulant added to it and served as the CMcontrol wells. The AE37 and control peptides and antigen were added inthe following amounts: 1.0 μg AEN, 10 μg AEN, 1.0 μg AE36, 10 μg AE36,1.0 μg AE37, 10 μg AE37, 0.2 μg TT. The PBMC population prepared fromthe patient's peripheral blood (as described in the previous section)was resuspended at 1.5×10⁶ cells/ml in CM and added as 200 μl aliquotsto the wells. The plate was then incubated in a humidified CO₂ incubatorfor a period of 4 days. On the third day of incubation the plate wasremoved and the wells were pulsed with 1 μCi/well of radioactive³H-thymidine after which the plate was returned to the incubator. On thefourth day the plate was removed from the incubator and the cells wereharvested onto a filtermat using a cell harvester (Harvester96-MachIII,Tomtec, Conn.). The filtermat was dried and placed in a sample bag withscintillation fluid (BetaScint, Perkin-Elmer) and counted using ascintillation counter (MicroBeta Trilux Scintillation Counter,Perkin-Elmer). The proliferative activity associated with the cultureswas measured by the amount of thymidine incorporation which wasdetermined as counts per minute (cpm). The average cpm was calculatedfor the triplicate cultures with the different peptides (FIGS. 15-18).The full range of concentrations and peptides tested and the number ofreplicate wells for each patient varied depending upon the availabilityof sufficient PBMC yield from the patient's blood sample.

Immunologic Response; Local Reaction; Delayed Type Hypersensitivity(DTH)

A direct in vivo measure of the vaccine's effectiveness is the DTHreaction. Patients were injected intradermally with 100 μg of AE37(without GM-CSF) prior to the initiation of the vaccination series andone month after the completion of the vaccination regimen in theopposite limb to which they received the vaccine series. Patients werealso injected intradermally with a parallel control (sterile saline,same volume) at a site on the back or extremity (opposite side from thevaccination site). The DTH reaction was measured in two dimensions at48-72 hours using the sensitive ballpoint pen method and compared withcontrol. Pre- and post-vaccination DTH reactions were also compared.

The mean diameter of local reaction pre vaccine was 4.9 mm (range 0-17mm) and the mean diameter post-vaccine was 62 mm (range 12-106 mm)(p<0.0001) (FIGS. 5-6). DTH did not correlate with other measures ofimmunologic response.

CONCLUSIONS

AE37 appears to be safe and well-tolerated with minimal systemictoxicity observed. Robust local reactions lead to dose reduction andeventual elimination of the immunoadjuvant GM-CSF. Despite noimmunoadjuvant, in vitro and in vivo immunologic responses weresignificant to both AE37 and the native AE36. To our knowledge, this isthe first report of a peptide vaccine derived from a tumor-associatedantigen being successful in eliciting an immunologic response in cancerpatients without an immunoadjuvant.

1. A method of treating a cancer in humans, the cancer beingcharacterized by expression of Her-2/neu, comprising: a) providing anIi-Key/MHC class II hybrid construct in a pharmaceutically acceptablecarrier, the construct comprising: i) the LRMK (SEQ ID NO: 2) amino acidresidues of li protein; and ii) an MHC class II epitope-containingsegment of Her-2/neu, directly or indirectly linked at its N-terminus toelement i); or DNA encoding the elements of i) and ii); and b)stimulating a patient's immune system by vaccination with the hybridconstruct of step a) under conditions appropriate for the stimulation ofan immune response.
 2. The method of claim 1 which is characterized bythe stimulation of CD4+ T cell production.
 3. The method of claim 1which is characterized by the stimulation of a CD4+ T cell responsespecific to native Her-2/neu peptide.
 4. The method of claim 1 whereinthe MHC Class II epitope is contained within the peptide GVGSPYVSRLLGICL(SEQ ID NO: 3).
 5. The method of claim 1 wherein the hybrid constructcomprises the peptide LRMKGVGSPYVSRLLGICL (SEQ ID NO: 4) or DNA encodingthe same.
 6. The method of claim 1 wherein the cancer is breast, ovary,recto-colon, lung, prostate, stomach, pancreatic, or biliary cancer. 7.The method of claim 1 wherein the cancer is breast cancer.
 8. Apharmaceutical composition for use in the treatment of cancer, thepharmaceutical composition comprising an Ii-Key/MHC Class II hybridconstruct in a pharmaceutically acceptable carrier, the Ii-Key/MHC ClassII hybrid construct comprising: a) the LRMK (SEQ ID NO: 2) residues ofthe Ii protein; and b) an MHC Class II epitope-containing segment ofHer-2/neu, directly or indirectly linked at its N-terminus to elementa); or DNA encoding the elements of a) and b).
 9. The pharmaceuticalcomposition of claim 8 further comprising an adjuvant.
 10. Thepharmaceutical composition of claim 9 wherein the adjuvant is GM-CSF.11. The pharmaceutical composition of claim 8 wherein the MHC Class IIepitope is contained within the peptide GVGSPYVSRLLGICL (SEQ ID NO: 3).12. The pharmaceutical composition of claim 8 comprisingLRMKGVGSPYVSRLLGICL (SEQ ID NO: 4) or a DNA encoding the same.
 13. Acomposition comprising: a) amino acid residues LRMK (SEQ ID NO: 2); andb) an MHC Class II epitope-containing segment of Her-2/neu, directly orindirectly linked at its N-terminus to element a); or DNA encoding theelements of a) and b).
 14. The composition of claim 13 wherein the MHCClass II epitope is contained within the peptide GVGSPYVSRLLGICL (SEQ IDNO: 3).
 15. The composition of claim 13 comprising LRMKGVGSPYVSRLLGICL(SEQ ID NO: 4) or a DNA encoding the same.